초록 ▼

Ⅲ. 연구개발의 내용 및 범위
제 2단계의 연구를 통해, 본 연구진은 다음과 같은 내용의 연구를 수행하였음.
< 1차년도 >
CRIF1에 의한 미토콘드리아 전사 조절 기전 규명
CRIF1에 의한 미토콘드리아 OXPHOS subunits assembly 조절 규명
CRIF1 conditional knockout를 위한 CRIF1 flox/flox mice line 확보
Brain specific CRIF1 knokout mice 생산 및 phenotype 분석
Muscle specific CRIF

Ⅲ. 연구개발의 내용 및 범위
제 2단계의 연구를 통해, 본 연구진은 다음과 같은 내용의 연구를 수행하였음.
< 1차년도 >
CRIF1에 의한 미토콘드리아 전사 조절 기전 규명
CRIF1에 의한 미토콘드리아 OXPHOS subunits assembly 조절 규명
CRIF1 conditional knockout를 위한 CRIF1 flox/flox mice line 확보
Brain specific CRIF1 knokout mice 생산 및 phenotype 분석
Muscle specific CRIF1 knokout mice 생산 및 phenotype 분석
< 2차년도 >
CRIF1에 의한 OXPHOS 구성 단백의 번역 조절 기전 규명
고지방식이에 따른 지방간에서의 CRIF1 결손모델에서 phenotype분석
Adipose tissue specific CRIF1 knockout mice 생산 및 phenotype 분석
Endothelial cell-specific CRIF1 knockout mice phenotype 분석
<3차년도 >
Islet specific CRIF1 knockout mice 생산 및 phenotype 분석
CRIF1응용 미토콘드리아 질환 치료용 모델개발
CRIF1응용 대사증후군 치료용 모델개발
인체질환모델에서 CRIF1 변이 및 발현이상 조사

Abstract ▼

We isolated a mitochondrial protein, called mtCRIF1, which is localized in inner membrane of mitochondria. In the subsequent studies, we found that mtCRIF1-deficient cells prepared from mtCRIF1 knockout mice showed marked deterioration of mitochondrial function resulted from loss of assembly in oxid

We isolated a mitochondrial protein, called mtCRIF1, which is localized in inner membrane of mitochondria. In the subsequent studies, we found that mtCRIF1-deficient cells prepared from mtCRIF1 knockout mice showed marked deterioration of mitochondrial function resulted from loss of assembly in oxidative phosphorylation (OXPHOS) I, III and IV complexes.
Here, we show that human or mouse cells lacking CRIF1 as a result of homologous recombination exhibit markedly impaired mitochondrial oxidative phosphorylation. CRIF1-deficient cells shows high lactate production and enhanced dependency on glycolytic ATP generation, due to severe reduction of respiratory chain complex activity. Although CRIF1 itself seems not a part of oxphos complex, CRIF1-deficient cells exhibit a reduced contents of complex I, III, IV and of its components, suggesting that CRIF1 plays key roles in the mitochondrial translation and/or the maintenance of mitochondrial oxphos supercomplex. These observations
suggested that CRIF1 deficiency may results mitochondrial diseasea in CRIF1-deficient animals. To verify this hypothesis, we are going to generate the tissue-specific knockout mice which showed specific deletion in Brain, islet (B-cells), skeletal muscle, adipose tissue and endothelial cells.
These brain-specific CRIF1 knockout animals exhibited locomotion disabilities at 12 weeks and showed markedly shortened life spans, with a maximum longevity of 24 weeks. Serial histological analysis from brain-specific CRIF1 knockout mice showed early characteristic changes of neuronal degeneration in hippocampus at 12 weeks. Beta cell-specific CRIF1 deficiency exhibited impaired beta cell functions and glucose intolerance. In addition, haploinsufficiency of CRIF1 resulted diabetogenic beta cell dysfunctions challenged on high fat diet. This beta cell-specific CRIF1 deficiency data provide novel mitochondrial function into the relationship between type 1 diabetes and type 2 diabetes. In liver-specific CRIF1-deficiency showed delayed glucose disappearance on glucose
challenge and insulin administration. In muscle specific CRIF1 knockout mice were approximately 3 months of age when they started developing a slowly progressive myopathy. Surprisingly, even though CRIF1 activity in CRIF1 KO muscles was <5% of control muscle at 2.5 months, these muscles were still able to contract at 80-100% of control maximal forces and showed only a 10% increase in fatigability, and no signs of oxidative damage or apoptosis were detected. The mouse model of adipocyte specific mitochondrial OXPHOS dysfunction by CRIF1 knockout showed loss of body weight and fat mass than control mouse. Also these mice had
smaller and dysplastic morphology of adipocyte in adipose tissue.
Consistently, CRIF1 depletion in 3T3-L1 fibroblast or ADSCs (Adipose derived stem cells) showed inhibition of mtDNA encoded OXPHOS subunit and differentiate into adipocyte with decrease of adipogenic gene expression. These results show mitochondrial OXPHOS is determine factor of adipocyte differentiation. Therefore, although mice haploinsufficient for CRIF1 in adipose tissue showed normal growth and development, they developed insulin resistance. CRIF1-silenced adipocytes showed higher expression of chemokines, the expression of which is dependent upon antioxidant and stress kinases. Accordingly, examination of adipose tissue
from CRIF1 haploinsufficient mice revealed increased expression of Mcp1, Ip10, Rantes and TNFα, as well as marked infiltration by macrophages.
These findings indicate that the OXPHOS status of adipose tissue determines its metabolic and inflammatory responses, and may cause systemic inflammation and insulin resistance. In summary, mitochondrial CRIF1 (mtCRIF1) is essential for the maintenance of normal mitochondrial oxphos functions and its dysregulation may be possibly linked to disorders related to cellular energy metabolism.

목차 Contents

• 표지 ... 1
• 제출문 ... 2
• 보고서 요약서 ... 3
• 요약문 ... 4
• SUMMARY ... 11
• CONTENTS ... 13
• 목차 ... 14
• 제1장 연구개발과제의 개요 ... 15
• 1. 연구개발의 목적 ... 15
• 제2장 국내외 기술개발 현황 ... 17
• 제3장 연구개발수행 내용 및 결과 ... 19
• 제1절 CRIF1에 의한 미토콘드리아 호흡복합체의 조절 ... 19
• 제2절 CRIF1에 의한 지방세포의 분화 ... 59
• 제3절 내피세포에서의 CRIF1 역할 ... 74
• 제4절 CRIF1에 의한 지방간 조절 ... 84
• 제5절 췌장 베타세포 특이적 CRIF1 결손 마우스 생산 및 표현형 분석 ... 94
• 제5절 근육 특이적 CRIF1 결손 마우스 생산 및 표현형 분석 ... 108
• 제6절 인체 질환 모델에서 CRIF1 돌연변이 조사 ... 114
• 제7절 인체 질환 모델에서 CRIF1발현이상 조사 ... 119
• 제4장 목표달성도 및 관련분야에의 기여도 ... 127
• (1) 연구개발의 최종목표 ... 127
• (2) 연차별 연구개발 목표 및 내용 ... 127
• (3) 계획대비 달성도 ... 128
• (4) 관련 분야의 기여도 ... 129
• (5) 위 연구목표(총연구기간)에서 중요도 순으로 4-5개 목표 추출 및 가중치 부여 ... 130
• 제5장 연구개발결과의 활용계획 ... 131
• 1. 추가연구의 필요성 ... 131
• 2. 타연구에의 응용 ... 131
• 제6장 연구개발과정에서 수집한 해외과학기술정보 ... 132
• 제7장 참고문헌 ... 135